• 미생물학회지
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• 제27권1호
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• pp.10-15
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• 1989

Mammalian cell 연구에 쓰기 위해 개발된 SV 40 transcriptional promoter를 함유하는 pKOneo plasmid를 발암 유전인자 연구에 쓰이는 NIH3T3 쥐 세포에 stable transfection 시켜 7개의 sub clones 얻었으며, 이 subclones이 갖는 세포 형질전환에 관한 여러가지 성질을 조사하였다. 실험결과에 따르면 stable transfection 후 세포 염색체에 삽입된 pKOneo plasmid 자체만으로도 NIH3T3 세포의 형질전환을 크게 일으키는 것으로 사료되었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권3호
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• pp.167-172
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• 2001

Staphylokinase (SAK) was produced in B. subtilis using two different promoter systems, i.e. the P43 and sacB promoters. To maximize SAK expression in B. subtilis, fermentation control strategies for each promoter were examined. SAK, under P43, a vegetative promoter transcribed mainly by $\sigma$(sup)B containing RNA polymerase, was overexpressed at low dissolved oxygen (D.O.) levels, suggesting that the sigB operon is somewhat affected by the energy charge of the cells. The expression of SAK at the 10% D.O. level was three times higher than that at the 50% D.O. level. In the case of sacB, a sucrose-inducible promoter, sucrose feeding was used to control the induction period and induction strength. Since sucrose is hydrolyzed by two sucrose hydrolyzing enzymes in the cell and culture broth, the control strategy was based on replenishing the loss of sucrose in the culture. With continuous feeding of sucrose, WB700 (pSAKBQ), which contains the SAK gene under sacB promoter, yielded ca. 35% more SAK than the batch culture. These results present efficient promoter-dependent control strategies in B. subtilis host system for foreign protein expression.

• Journal of Korean Neurosurgical Society
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• 제46권4호
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• pp.385-388
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• 2009

Objective : We analyzed the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter in World Health Organization (WHO) grade III gliomas in association with other molecular markers to evaluate their prevalence. Methods : The samples of a total of 36 newly WHO grade III glioma patients including 19 anaplastic oligodendrogliomas (AO), 7 anaplastic oligoastrocytomas (AOA), and 10 anaplastic astrocytomas (AA) were analyzed. The methylation status of the MGMT gene promoter was confirmed by methylation-specific polymerase chain reaction. The 1p/19q chromosomal deletion status and EGFR amplification were assessed by Fluorescence In-Situ Hybridization. MGMT, EGFR, EGFRvlll, and p53 expression were analyzed by immunohistochemical staining. Results : The MGMT gene promoter was methylated in 32 (88.9%) and unmethylated in 4 (11.2%) Among them, all of the AO and AOA had methylated MGMT gene promoter without exception. Significant associations between MGMT gene promoter hypermethylation and 1p/19q deletion was observed (p=0.003). Other molecular markers failed to show significant associations between MGMT gene promoter statuses. Conclusion : There was extensive epigenetic silencing of MGMT gene in high grade gliomas with oligodendroglial component. Together with frequent 1p/19q co-deletion in oligodendroglial tumors, this may add plausible explanations supporting the relative favorable prognosis in oligodendroglial tumors compared with pure astrocytic tumors.

• 한국식품위생안전성학회지
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• 제6권3호
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• pp.133-137
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• 1991

5'-XMP를 5'-GMP로 전환하는 효소인 XMP aminase[EC 6.3.4.1]의 활성을 증가시키기 위하여 XMP aminase의 유전자를 함유한 1.7kb gua A gene fragment를 pLC 34-10으로부터 분리하여 pBR 322에 subcloning 한 뒤 trp promoter를 가지고 있는 대장균 발현 벨터 pDR 720에 도입하였다. 재조합된 pXAR 64에 존재하는 gua A 유전자는 trp Promoter에 의하여 발현이 증대되었으며 $3-{\beta}-indoleacrylic$ acid에 의하여 XMP aminase의 생성이 유도되었다. XMP aminase의 비활성은 pLC 34-10을 함유한 균주에 비하여 약 17배 증가되었다.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.337-342
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• 2019

We published that bacterial heme was over-produced in a recombinant Corynebacterium glutamicum expressing 5-aminolevulinic acid synthase ($hemA^+$) under control of a constitutive promoter ($P_{180}$) and the heme-producing C. glutamicum had commercial potentials; as an iron feed additive for swine and as a preservative for lactic acid bacteria. To enhance the heme production, the $hemA^+$ gene was expressed under controls of various promoters in the recombinant C. glutamicum. The $hemA^+$ expression by $P_{gapA}$ (a constitutive glycolytic promoter of glyceraldehyde-3-phosphate dehydrogenase) led 75% increase of heme production while the expression by $P_{H36}$ (a constitutive, very strong synthetic promoter) resulted in 50% decrease compared with the control ($hemA^+$ expression by $P_{180}$ constitutive promoter). The $hemA^+$ expression by a late log-phase activating $P_{sod}$ (an oxidative-stress responding promoter of superoxide dismutase) led 50% greater heme production than the control. The $hemA^+$ expression led by a heat-shock responding chaperone promoter ($P_{dnaK}$) resulted in 121% increase of heme production at the optimized heat-shock conditions. The promoter strength and induction phase are discussed based on the results for the heme production at an industrial scale.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.240-245
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• 1991

We have constructed a promoter-probe vector pKU20 using pKT230, a derivative of broad-host-range plsmid RSF1010, as a base. The pKU20 contains structural gene for aminoglycoside phos-photransferase (aph), without promoter, and a multiple cloning site upstream the aph. Using this vector, a 412base pairs (bp) PstI fragment showing strong promoter activity both in Escherichia coli LE392 and Pseudomonas putida KCTC1644 has been cloned from Pseudomonas fluorescens chromosomal DNA on the basis of streptomycin resistance. The nucleotide sequence of the 412 bp fragment has been determined and the putative - 35 and -10 region was observed. Insecticidal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 inserted on downstream of the promoterlike DNA fragment was efficiently expressed in E. coli and P. putida. The toxin protein was efficiently synthesized in an insoluble form in both strains.

• Tuberculosis and Respiratory Diseases
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• 제73권1호
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• pp.11-21
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• 2012

Background: While qualitative analysis of methylation has been reviewed, the quantitative analysis of methylation has rarely been studied. We evaluated the methylation status of CDKN2A, $RAR{\beta}$, and RASSF1A promoter regions in non-small cell lung carcinomas (NSCLCs) by using pyrosequencing. Then, we evaluated the association between methylation at the promoter regions of these tumor suppressor genes and the clinicopathological parameters of the NSCLCs. Methods: We collected tumor tissues from a total of 53 patients with NSCLCs and analyzed the methylation level of the CDKN2A, $RAR{\beta}$, and RASSF1A promoter regions by using pyrosequencing. In addition, we investigated the correlation between the hypermethylation of CDKN2A and the loss of $p16^{INK4A}$ immunoexpression. Results: Hypermethylation of CDKN2A, $RAR{\beta}$, and RASSF1A promoter regions were 16 (30.2%), 22 (41.5%), and 21 tumors (39.6%), respectively. The incidence of hypermethylation at the CDKN2A promoter in the tumors was higher in undifferentiated large cell carcinomas than in other subtypes (p=0.002). Hyperrmethylation of CDKN2A was significantly associated with $p16^{INK4A}$ immunoexpression loss (p=0.045). With regard to the clinicopathological characteristics of NSCLC, certain histopathological subtypes were found to be strongly associated with the loss of $p16^{INK4A}$ immunoexpression (p=0.016). Squamous cell carcinoma and undifferentiated large cell carcinoma showed $p16^{INK4A}$ immunoexpression loss more frequently. The Kaplan-Meier survival curves analysis showed that methylation level and patient survival were barely related to one another. Conclusion: We quantitatively analyzed the promoter methylation status by using pyrosequencing. We showed a significant correlation between CDKN2A hypermethylation and $p16^{INK4A}$ immunoexpression loss.

• Asian Pacific Journal of Cancer Prevention
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• 제16권16호
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• pp.7111-7115
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• 2015

Background: The aim of the meta-analysis was to derive a more precise assessment of the association between p16 promoter methylation and thyroid cancer risk. Materials and Methods: The PubMed, Web of Science databases and Chinese CNKI were searched for relevant articles. Ultimately, seventeen case-control studies were included with a total of 804 thyroid cancer cases and 487 controls analysis by R Software (R version 3.1.2) including meta. Crude odds ratios with 95% confidence intervals were calculated using the random-effects model which were used to assess the strength of relationship between p16 methylation and lung carcinogenesis. Funnel plots were carried out to evaluate publication bias. Results: The meta-analysis results showed that the frequency of p16 promoter methylation in cancer tissue/blood was significantly higher than that normal tissue/blood (OR=5.46, 95%CI 3.12-9.55, P<0.0001) by random effects model with small heterogeneity. Conclusions: Thus, p16 promoter methylation may be associated with thyroid cancer risk.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.551-553
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• 2000

A strong constitutive $P_{JH}$ promoter from Bacillus was applied to overexpress the endoxylanase gene in B. subtilis. The expression plasmid, pHJKJ4, was designed to contain the $P_{JH}$ promoter and endoxylanase promoter ($P_B$), and introduced into B. subtilis DB104. Through batch fermentation of the trasformant cell on a maltose medium, endoxylanase was produced in a growth-associated manner as the predominant protein. The total activity reached about 600 unit/ml at the end of the cultivation, which corresponded to 698 mg endoxylanase protein/l with a specific activity of 860 unit/mg protein. It was also found that the segregational plasmid instability was less than 30% and most of the endoxylanase activity was detected in the culture medium. This result suggests that the secretory production of endoxylanase can be significantly enhanced with the use of the $P_{JH}$ promoter and high-cell density culture techniques, quantitatively as well as qualitatively.

• Journal of Applied Biological Chemistry
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• 제53권1호
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• pp.1-7
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• 2010

xylA 프로모터는 대장균의 xylose 대사에 관여하는 xylose 오페론 상의 중요한 프로모터이다. 이 프로모터는 xylose에 의해 강하게 조절을 받는다고 알려져 있다. 이러한 특징은 새로운 발현 백터를 구축하는데 충분한 조건을 갖추고 있다고 생각된다. 본 연구에서는 이러한 xylose에 의해 유도 되는 발현벡터를 구축하기 위하여 600 bp의 xylA 프로모터를 증폭하여 pUC18의 AatII와 HindIII 사이에 삽입하여 pXA600을 구축하였다. 또한 조절단백질인 XylR의 영향을 조사하기 위하여 xylR 유전자를 삽입하여 pXAR600을 구축하였다. 발현의 강도를 측정하기 위하여 3,048 bp의 lacZ유전자를 xylA 프로모터의 하류에 연결하여 pXA600-lacZ와 pXAR600-lacZ를 구축하고 대장균 JM109에 형질전환시켰다. 구축된 pXA600-lacZ와 pXAR600-lacZ는 LB 배지에서 배양하였을 때 xylose 유도하에서 각각 1,641 unit와 2,304 unit의 $\beta$-galactosidase 활성을 보였으며, DM 배지상에서 배양했을 때 xylose 유도 시 각각 6,282 unit와 9,320 unit의 $\beta$-galactosidase 활성을 보였다. 또한 왜래 유전자의 발현 가능성을 확인하기 위하여 S. thermocyaneoviolaceus의 내열성 xylanase를 코딩하는 xynA 유전자를 실제로 구축된 pXA600과 pXAR600에서 발현을 확인하여 pXA600 및 pXAR600이 새로운 xylose 유도성 발현벡터로서의 사용 가능성을 확인하였다.